TAILIEUCHUNG - Báo cáo khoa học: Characterization of the secreted chorismate mutase from the pathogen Mycobacterium tuberculosis

The gene encompassing ORF Rv1885c with weak sequence similarity to AroQ chorismate mutases (CMs) was cloned from the genome ofMycobac-terium tuberculosis and expressed in Escherichia coli. The gene product (*MtCM) complements a CM-deficient E. colistrain, but only if produced without the predicted N-terminal signal sequence typical ofM. tuberculosis. The mature *MtCM, which was purified by exploiting its resistance to irre-versible thermal denaturation, possesses high CM activity in vitro. The enzyme follows simple Michaelis–Menten kinetics, having a kcat of 50 s )1 and aKmof 180lm(at 30 C and pH ) | ềFEBS Journal Characterization of the secreted chorismate mutase from the pathogen Mycobacterium tuberculosis Severin Sasso Chandra Ramakrishnan Marianne Gamper Donald Hilvert and Peter Kast Laboratorium fur Organische Chemie Swiss Federallnstitute of Technology Zurich ETH Switzerland Keywords chorismate mutase Mycobacterium tuberculosis pathogenesis shikimate pathway signal sequence Correspondence P. Kast Laboratorium fur Organische Chemie Swiss Federallnstitute of Technology ETH Honggerberg - HCI F333 CH-8093 Zurich Switzerland Fax 41 1633 1326 Tel 41 1632 2908 E-mail kast@ Received 12 October 2004 revised 4 November 2004 accepted 1 2 November 2004 doi The gene encompassing ORF Rv1885c with weak sequence similarity to AroQ chorismate mutases CMs was cloned from the genome of Mycobacterium tuberculosis and expressed in Escherichia coli. The gene product MtCM complements a CM-deficient E. coli strain but only if produced without the predicted N-terminal signal sequence typical of M. tuberculosis. The mature MtCM which was purified by exploiting its resistance to irreversible thermal denaturation possesses high CM activity in vitro. The enzyme follows simple Michaelis-Menten kinetics having a kcat of 50 s-1 and a Km of 180 pM at 30 C and pH . MtCM was shown to be a dimer by analytical ultracentrifugation and size-exclusion chromatography. Secondary-structure prediction and CD spectroscopy confirmed that MtCM is a member of the all-a-helical AroQ class of CMs but it seems to have a topologically rearranged AroQ fold. Because CMs are normally intracellular metabolic enzymes required for the biosynthesis of phenylalanine and tyrosine the existence of an exported CM in Gram-positive M. tuberculosis is puzzling. The observation that homologs of MtCM with a predicted export sequence are generally only present in parasitic or pathogenic organisms suggests that secreted CMs may have evolved to participate in some aspect .

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